Vacuum-assisted plasma separation

ABSTRACT

A plasma separation system and process for providing filtered plasma from a blood sample is described. The system may include a blood separation well having a separation membrane for filtering the blood sample. The filtering process may be aided by the use of a negative or positive pressure source attached to the plasma separation system.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE

The present patent application is a divisional of U.S. Ser. No.15/329,795, filed on Jan. 27, 2017; which is a national stage entry fromthe Patent Cooperation Treaty Application identified by serial numberPCT/US2015/042838, publication number, WO 2016/019113, titled“Vacuum-Assisted Plasma Separation”, filed on Jul. 30, 2015; whichclaims benefit under 35 USC § 119(e) of U.S. Provisional Application No.62/031,908, filed Aug. 1, 2014. The entire contents of theabove-referenced patent applications are hereby expressly incorporatedherein by reference.

BACKGROUND

Plasma, rather than whole blood, is generally the preferred sample formany clinical diagnostic tests. For example, in HIV viral loaddetection, plasma is separated from whole blood as hemoglobin and otherhemolysis products may interfere with detection of viral RNA. Ashemoglobin and other hemolysis products may interfere with assayresults, plasma may need to be non-hemolyzed.

Within the industry, plasma is usually obtained by centrifuging wholeblood separating red blood cells from plasma. The centrifuging process,however, may be slow and require large powered instrumentation.Additionally, once the centrifugation process has begun, the instrumentis unavailable for use by another operator until completion of theprocess.

Progress within the medical industry has been in the development ofpoint-of-care systems to provide rapid and portable care. Because oftime restraints, size of equipment, and one-operator use, centrifugationis generally impractical for use in such point-of-care diagnosticinstruments. Other separation methods including laminar-flow filtrationor capillary-action based processes, however, are also expensive,complex, slow, require large volumes of blood, or may lead tounacceptable levels of hemolysis.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

To assist those of ordinary skill in the relevant art in making andusing the subject matter hereof, reference is made to the appendeddrawings, which are not intended to be drawn to scale, and in which likereference numerals are intended to refer to similar elements forconsistency. For purposes of clarity, not every component may be labeledin every drawing.

FIG. 1 is an exploded perspective view of an exemplary plasma separationsystem in accordance with the present disclosure.

FIG. 2A is a cross-sectional view of the plasma separation system ofFIG. 1, taken along the lines 2A-2A in FIG. 1.

FIG. 2B is a cross-sectional view of an exemplary filter for use in theplasma separation system of FIG. 1, taken along the lines 2B-2B in FIG.1.

FIG. 2C is a sectional view of an exemplary separation membrane for usein the plasma separation system of FIG. 1, taken along the lines 2C-2Cin FIG. 1.

FIG. 2D is a sectional view of an exemplary adhesive member for use inthe plasma separation system of FIG. 1, taken along the lines 2D-2D inFIG. 1.

FIG. 3 is a partial top down view of the plasma separation system ofFIG. 1 having the filter, the separation membrane, and the adhesivemember removed.

FIG. 4 is a partial perspective view of another version of an exemplaryplasma separation system having a channel connected to a proximal end ofa plasma collection vessel in accordance with the present disclosure.

FIG. 5 is a top down view of yet another version of an exemplary plasmaseparation system having a plasma collection vessel with a serpentinechannel in accordance with the present disclosure.

FIG. 6 is a flow chart of an exemplary method for separating filteredplasma from a blood sample in accordance with the present disclosure.

FIGS. 7A-7C collectively illustrate the use of an exemplary plasmaseparation system for separating filtered plasma from a blood sample inaccordance with the method of FIG. 6.

FIG. 8 is a cross-sectional view of another version of an exemplaryplasma separation system having a pressure system connected to a bloodseparation well in accordance with the present disclosure

DETAILED DESCRIPTION

Before explaining at least one embodiment of the disclosure in detail,it is to be understood that the disclosure is not limited in itsapplication to the details of construction, experiments, exemplary data,and/or the arrangement of the components set forth in the followingdescription or illustrated in the drawings unless otherwise noted.

The disclosure is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for purposes ofdescription, and should not be regarded as limiting.

The following detailed description refers to the accompanying drawings.The same reference numbers in different drawings may identify the sameor similar elements.

As used in the description herein, the terms “comprises,” “comprising,”“includes,” “including,” “has,” “having,” or any other variationsthereof, are intended to cover a non-exclusive inclusion. For example,unless otherwise noted, a process, method, article, or apparatus thatcomprises a list of elements is not necessarily limited to only thoseelements, but may also include other elements not expressly listed orinherent to such process, method, article, or apparatus.

Further, unless expressly stated to the contrary, “or” refers to aninclusive and not to an exclusive “or”. For example, a condition A or Bis satisfied by one of the following: A is true (or present) and B isfalse (or not present), A is false (or not present) and B is true (orpresent), and both A and B are true (or present).

In addition, use of the “a” or “an” are employed to describe elementsand components of the embodiments herein. This is done merely forconvenience and to give a general sense of the inventive concept. Thisdescription should be read to include one or more, and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.Further, use of the term “plurality” is meant to convey “more than one”unless expressly stated to the contrary.

As used herein, any reference to “one embodiment,” “an embodiment,”“some embodiments,” “one example,” “for example,” or “an example” meansthat a particular element, feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. The appearance of the phrase “in some embodiments” or “oneexample” in various places in the specification is not necessarily allreferring to the same embodiment, for example.

Referring now to the Figures, and in particular to FIG. 1, shown thereinand designated by reference numeral 10 is an exemplary plasma separationsystem 10 in accordance with the present disclosure. Generally, theplasma separation system 10 may provide separation of plasma from bloodusing a vacuum force, and without using centrifugation or laminar-flowfiltration based filtration processes. The separation of plasma fromblood may be with minimal hemolysis and within a relatively short amountof time (e.g., 10-90 seconds) as compared to processes used currentlywithin the industry. In some embodiments, the plasma may be further usedin one or more point-of-care assays.

The plasma separation system 10 may include a housing 12 supporting orencompassing a blood separation well 14 connected via a first channel 16to a plasma collection vessel 18. The housing 12 supports or encompassesa second channel 22 connecting the plasma collection vessel 18 to anoutlet port 24 downstream of the plasma collection vessel 18. The plasmaseparation system 10 may also include a negative pressure source 26 thatcan be attached to the outlet port 24. In this instance, the outlet port24 can be configured to allow for the negative pressure source 26 to beattached thereto assist in enabling the operation of the plasmaseparation system 10.

Generally, in the plasma separation system 10, blood may be added to theblood separation well 14. Negative pressure (e.g., vacuum pressure) maybe applied by the negative pressure source 26 to the outlet port 24,which also causes a vacuum to form in the blood separation well 14.Using a combination of capillary action and negative pressure, plasmamay be separated from the blood. The magnitude of the negative pressuremay be controlled to prevent hemolysis and/or leakage of cellularmaterial. The separated plasma may be collected in the plasma collectionvessel 18.

In some embodiments, the plasma separation system 10 may be a single-usesystem. Alternatively, one or more components of the plasma separationsystem 10 may be disposable such that the plasma separation system 10may be a multi-use system. For example, in some embodiments, after afirst blood sample is separated, one or more channels 16 and 22 may belined such that the plasma collection vessel 18 and components of theblood separation well 14 may be removed, disposed of, and replaced foruse with a second blood sample.

The housing 12 may be formed of materials including, but not limited to,glass, plastic, and/or the like. The shape and size of the housing 12may be dependent on shape and/or size of the blood separation well 14,channels 16 and 22, and/or the plasma collection vessel 18. The housing12 that is shown in FIG. 1 is a unitary integral device that is shapedto form the blood separation well 14, channels 16 and 22 and the plasmacollection vessel 18. It should be understood that the housing 12 can beconstructed of separate components which are connected together so thatthe blood separation well 14, channels 16 and 22 and the plasmacollection vessel 18 communicate with each other. Generally, the size ofthe housing 12 may be minimized and determinate on an amount of plasmathat is desired (e.g., 10-20 μL) to be extracted from a blood sample.The housing 12 may include a surface 28.

The blood separation well 14 may be positioned to intersect the surface28 of the housing 12 such that the surface 28 at least partiallysurrounds the blood separation well 14. For example, in someembodiments, the blood separation well 14 may include a recess 30intersecting the surface 28 of the housing 12 as illustrated in FIGS. 1and 2 and in this instance the surface 28 may form a rim surrounding therecess 30. The recess 30 may include a proximal end 32 and a distal end34 with a wall 36 spanning the length of the proximal end 32 to thedistal end 34. The proximal end 32 may include a capillary surface 38 inwhich microchannels are formed in the proximal end 32 as discussed inmore detail below with respect to FIG. 3.

The plasma separation device 10 may also be provided with a filter 40, aseparation membrane 42, and an adhesive member 44. The filter 40, theseparation membrane 42 and the adhesive member 44 may be stacked on thecapillary surface 38 and disposed within the recess 30 such that thefilter 40 is below the surface 28 of the housing 12 and fully disposedwithin the recess 30. For example, in some embodiments, the adhesivemember 44 may be positioned on the capillary surface 38 with theseparation membrane 42 and the filter 40 positioned thereon,respectively. In some embodiments, the filter 40 and/or separationmembrane 42 may be pressure fit (also known as a “press fit”) within therecess 30.

The size and shape of the recess 30 may be dependent on the size andshape of the filter 40, the separation membrane 42, and/or the adhesivemember 44. For example, in some embodiments, the size and shape of therecess 30 may be circular as the filter 40, separation membrane 42, andadhesive member 44 are circular. The shape, however, may be any shapeincluding, but not limited to, rectangular, triangular, or any fancifulshape. The volume of the recess 30, including the height and/or lengthof wall 36 may be dependent on thicknesses and/or the widths of one ormore of the filter 40, separation membrane 42, and/or adhesive member44. Generally, the filter 40, separation membrane 42 and the adhesivemember 44 may be positioned within the recess 30 such that each remainsbetween the proximal end 32 and the distal end 34 of the recess 30.Further, although the wall 36 is depicted as a straight wall, it shouldbe understood that in some embodiments, the wall 36 can be stepped.

In some embodiments, one or more additional layers may be positioned ona first side 48 of the filter 40 to hold the filter 40 and separationmembrane 42 within the blood separation well 14. For example, in oneexample, a plastic O-ring may be positioned on the first surface 48 ofthe filter 40 to hold the filter 40 and separation membrane 42 withinthe blood separation well 14. In another example, a cap (e.g., plasticformed cap) may be positioned adjacent or proximal to the first side 48of the filter 40 and span the length of the blood separation well 14. Insome embodiments, the cap may be vented to allow for escape of gas tothe outside environment. The cap may serve to hold the filter 40 inclose proximity to the separation membrane 42. In one example, as bloodwicks into the filter 40 during use, the filter 40 may expand in sizesuch that the cap retains the filter 40 within the blood separation well14. To that end, the filter 40 may expand in the direction of theseparation membrane 42 positioning the filter 40 in close proximity oreven in contact with the separation membrane 42.

The filter 40 may be formed of one or more layers 46. Each layer 46 mayinclude the first side 48 and a second side 50 generally opposite of thefirst side 48. Generally, blood is provided onto and contacts the firstside 48 of the filter 40 and passes through the layer 46 of the filter40 such that filtered blood emerges on the second side 50. The filteredblood may include plasma and a portion of red blood cells that managedto pass through the layer 46. Some of the red blood cells will becaptured within the layer 46 of the filter 40.

In other words, the filter 40 generally removes a portion of the redblood cells from the blood and also may reduce separation burden of theseparation membrane 42. For example, the filter 40 may remove red bloodcells (e.g., up to 70% of red blood cells) within the blood sample. Theremoval of a significant portion of red blood cells may aid the flow ofthe filtered blood through the separation membrane 42 and also reduceclogging of the separation membrane 42.

Each layer 46 may be formed of materials including, but not limited to,glass fibers, polyester fibers, cellulose fibers, and/or the like. Forexample, one or more filters 40 may be a commercially available filterunder the trade name designations of VF1, VF2 and GFB, manufactured anddistributed by Whatman, having a location in Maidstone Kent. Forexample, in some embodiments, one or more filters 40 may be a 9.5 mmdisc of Whatman VF2 glass fiber.

The second side 50 of the filter 40 may be in proximity to or contactthe separation membrane 42. Generally, the separation membrane 42 mayremove the remaining red blood cells from the filtered blood providingfiltered plasma. The filtered plasma may be essentially cell-free inthat the filtered plasma includes minimal cellular debris. For example,hemoglobin content with the filtered plasma may be comparable to plasmaobtained by centrifuge techniques currently known within the industry.

In some embodiments, the plasma separation system 10 may solely comprisethe separation membrane 42 without the use of the filter 40. Forexample, the blood may be filtered solely through the separationmembrane 42 providing filtered plasma using the methods as describedherein.

The separation membrane 42 may be formed of one or more layers 52. Eachlayer 52 may have a first side 54 and a second side 56. Generally, thefiltered blood contacts the first side 54 of the separation membrane 42passing through the layer 52 of the separation membrane such thatfiltered plasma emerges on the second side 56.

The separation membrane 42 may be formed of materials including, but notlimited to, nylon, polysulfone, polycarbonate and/or the like. Forexample, in some embodiments, the separation membrane 42 may be anion-tracked etched membrane.

In some embodiments, the separation membrane 42 may be an asymmetricmembrane. For example, the separation membrane 42 may be formed havingat least a first set of pores and a second set of pores, with the firstset of pores and the second set of pores being different sizes.Generally, larger pores may be formed on the first side 54 of theseparation membrane 42 and smaller pores may be formed on the secondside 56 of the separation membrane 42 such that the filtered blood flowsthrough the larger pores to the small pores. This may reduce blockage ofred blood cells within the separation membrane 42.

In some embodiments, one or both of the filter 40 and/or separationmembrane 42 may be treated with one or more blocking agents and/orsurfactants. Treatment with blocking agents and/or surfactant mayenhance analyte recovery and/or plasma separation efficiency.Surfactants may include, but are not limited to, Tween-20, and/or thelike.

By treating with one or more blocking agents, the filter 40 and/orseparation membrane 42 may be made more or less hydrophilic, more orless hydrophobic, more or less susceptible to protein adsorption, moreor less positively charged, more or less negatively charged and/or thelike. For example, in some embodiments, the filter 40 and/or separationmembrane 42 may be treated with PAMAM dendrimers, Merquat, or otherpolycations. Blocking agents may include, but are not limited to bovineserum albumin, Seablock, gelatin, and/or the like.

The second surface 56 of the separation membrane 42 may contact theadhesive member 44. In some embodiments, the adhesive member 44 mayprevent leakage of blood cells around the perimeter of the separationmembrane 42.

The adhesive member 44 may include a first surface 58 and a secondsurface 60 with the first surface 58 contacting the second surface 56 ofthe separation membrane 42 and the second surface 60 adhered to at leasta portion of the capillary surface 38 of the recess 30. Generally, theadhesive member 44 aids in holding the separation membrane 42 within therecess 30. To that end, in some embodiments, each surface 58 and 60 ofthe adhesive member 44 may include an adhesive material. Adhesivematerial may include, but is not limited to, polyethylene terephthalate(PET) with silicone adhesive, and/or the like. For example, the adhesivemember 44 may be a double-sided PET adhesive O-ring as illustrated inFIGS. 2A and 2D. In some embodiments, the adhesive member 44 may beintegral to the blood separation well 14 or adhered to the wall 36 ofthe recess 30.

Size and shape of the adhesive member 44 may be dependent on the sizeand shape of the separation membrane 42 such that the separationmembrane 42 may be positioned within the blood separation well 14 andleakage of filtered blood about edges of the separation membrane 42 maybe minimized or eliminated.

The size and shape of the adhesive member 44 may be determined such thatthe separation membrane 42 is placed in close contact with the capillarysurface 38 of the recess 30 of the blood separation well 14 whilepreventing leakage of blood within the blood separation well 14. In someembodiments, the shape of the adhesive member 44 may include an opening62. The opening 62 may provide for flow of the filtered plasma to flowfrom the second surface 56 of the separation membrane 42 to thecapillary surface 38 of the recess 30. For example, in some embodiments,the adhesive member 44 may be formed as an O-ring, or any fanciful shapeproviding a direct opening 62 for flow of filtered plasma from thesecond surface 56 of the separation membrane 42 to the capillary surface38 of the recess 30. Additionally, in some embodiments, the material ofthe adhesive member 44 may be formed of a mesh-type material.

Referring to FIGS. 2A and 3, the capillary surface 38 of the recess 30of the blood separation well 14 may include one or more microchannels64. Microchannels 64 may encourage capillary flow of the filteredplasma. Microchannels 64 may form any pattern capable of enhancingcapillary flow of the filtered plasma through the blood separation well14. For example, in FIGS. 1 and 3, eight microchannels 64 are used toform a concentric pattern having a plurality of radial microchannelsconnecting at a central axis 66. Although eight microchannels 64 areillustrated in FIGS. 1 and 3, it should be apparent to one skilled inthe art that any number of microchannels 64 may be used so long as suchmicrochannels 64 are positioned within the confines of the capillarysurface 38. Additionally, one or more tributaries may be included withinthe concentric pattern. It should be noted that the capillary surface38, in some embodiments, may not include microchannels 64 as capillaryflow may still occur without such microchannels 64.

In some embodiments, one or more venting channels 68 may be positionedwithin the recess 30 of the blood separation well 14. For example, inFIGS. 1 and 3, four venting channels 68 are provided within the recess30 of the blood separation well 14 extending from the capillary surface38 to the surface 28. Venting channels 68 may provide for venting of gas(e.g., air) within the blood separation well 14. For example, gasentering the filter 40 and/or separation membrane 42 may exit the bloodseparation well 14 through the one or more venting channels 68.

The filtered plasma may flow from the blood separation well 14 to thechannel 16. The channel 16 may include a first outlet 70 connected tothe plasma collection vessel 18. In some embodiments, the channel 16 mayalso include a second outlet 72. Generally, the second outlet 72 may beblocked during operation of the plasma separation system 10 to cause thevacuum force to be directed into the blood separation well via thechannel 16. The second outlet 72 may be capable of being selectivelyopened to provide for removal of any additional filtered blood and/orfiltered plasma from within the blood separation well 14 and/or channel16.

The filtered plasma may flow through the channel 16 and through thefirst outlet 70 to the plasma collection vessel 18. The plasmacollection vessel 18 may have a proximal end 74 and a distal end 76connected by a tapered wall 78. For example, the width of the plasmacollection vessel 18 may increase from the proximal end 74 to the distalend 76. Although the shape of the plasma collection vessel 18 is shownas conical, it should be apparent that the plasma collection vessel 18may be formed in other shapes (e.g., cylindrical). Generally, the plasmacollection vessel 18 may be formed such that the amount of volume wherethe filtered plasma collects reduces dead volume. For example, the shapeof the plasma collection vessel 18 may be formed such that the filteredplasma collects in an area wherein recovery of the filtered plasma by apipette or other collection means may be maximized (e.g., collection of10-20 μL of filtered plasma).

Generally, the first outlet 70 may be positioned below the output port24 and near the proximal end 74 of the plasma collection vessel 18.Collection of filtered plasma may be in a portion 79 positioned belowthe first outlet 70, e.g., between the first outlet 70 and the proximalend 74 of the plasma collection vessel 18 as illustrated in FIG. 2A.

The plasma collection vessel 18 may include a seal 80. The seal 80 maycover the distal end 76 of the plasma collection vessel 18 so thatvacuum force applied to the output port 24 of the channel 22 is directedthrough the plasma collection vessel 18 and into the channel 16. In someembodiments, the seal 80 may be formed of pierceable material. Forexample, the seal 80 may be formed of materials capable of being piercedby a pipet tip or similar mechanism for collection of plasma from theplasma collection vessel 18. Such materials may include, but are notlimited to, acetate, polyethylene, foil and/or the like.

The plasma collection vessel 18 may be connected to the channel 22. Thechannel 22 may be positioned above the channel 16, and in proximity tothe distal end 76 of the plasma collection vessel 18, e.g., between thechannel 16 and the distal end 76. The channel 22 may connect the plasmacollection vessel 18 to the negative pressure source 26 via the outletport 24. In some embodiments, the outlet port 24 of the channel 22 mayinclude a fitted tube or luer connection at which the negative pressuresource 26 is connected.

The negative pressure source 26 may be any source capable of providingforce between approximately 0.05-2 psi. For example, the negativepressure source 26 may include, but is not limited to, a pump, a syringepump, a vacuum pump, a suction pump, and/or the like. Generally, thenegative pressure source 26 may be capable of being controlled such thatplasma may be collected without hemolysis and/or free of cellularmaterial from the blood. For example, by controlling the force of thenegative pressure source 26 within the boundaries discussed above, therisks of damage to the plasma may be reduced such that the blood may nothemolyze and/or cells may not deform (i.e., pass through the separationmembrane 42).

Control of the negative pressure source 26 may provide for a fixed flowrate and volume. For example, in some embodiments, one or more pressuresensors or monitors may be used to control the negative pressure source26. In another example, displacement speed of a syringe may control therate and volume of the negative pressure source 26.

FIG. 4 illustrates another exemplary embodiment of a plasma separationsystem 10 a. Similar to the plasma separator system 10 of FIG. 1, theplasma separation system 10 a includes the housing 12 supporting orencompassing the blood separation well 14. The blood separation well 14is connected via a first channel 16 a to a plasma collection vessel 18 asuch that filtered plasma enters the plasma collection vessel 18 a froma proximal end 94 of the plasma collection vessel 18 a. A second channel22 connects the plasma collection vessel 18 a to an outlet port 24downstream of the plasma collection vessel 18 a. The outlet port 24 mayallow for a negative pressure source 26 (e.g., vacuum source) to beattached to the plasma separator system 10 a.

The channel 16 a may be implemented in a variety of manners such thatthe channel 16 a connects the plasma collection vessel 18 a to theproximal end 94 of the plasma collection vessel 18 a. For example, thechannel 16 a may include a variety of linear segments that areinterconnected as shown in FIG. 4. In the example depicted in FIG. 4,the channel 16 a includes a first portion 82 and a second portion 84that may be in parallel alignment connected by a third portion 86extending between the first portion 82 and the second portion 84. In theexample shown, the third portion 86 extends normally to the firstportion 82 and the second portion 84 and vertically within the housing12. Although the channel 16 a includes corners 88 and 90 formed byintersection of the first portion 82 and the second portion 84 with thethird portion 86, it should be noted, the corners 88 may include roundededges. Additionally, the third portion 86 may be positioned at an anglerelative to the first portion 82 and the second portion 84 such that thethird portion 86 provides a sloped connection between the first portion82 and the second portion 84. A fourth portion 92 of the channel 16 amay provide an inlet 94 into the proximal end 94 of the plasmacollection vessel 18 a.

Generally, in the plasma separation system 10 a, blood may be added tothe blood separation well 14. Vacuum pressure may be applied via theoutlet port 24. Using a combination of capillary action and vacuumpressure, plasma may be separated from the blood. The plasma may enterand collect at the proximal end 94 of the plasma collection vessel 18 a.The vacuum pressure may be controlled to prevent hemolysis and/orleakage of cellular material.

FIG. 5 illustrates another exemplary embodiment of a plasma separationsystem 10 b which is similar in construction to the plasma separationsystems 10 and 10 a shown in FIGS. 1 and 4 with the exception that thatplasma separation system 10 b includes a plasma collection vessel 18 bin the form of a serpentine channel 96, rather than a well. Since thevolume of the serpentine may be controlled by the length of theserpentine channel 96, the system 10 b can be used to meter the plasma.The serpentine channel 96 can be readily integrated with other standardmicrofluidic features such as valves and reaction wells for performingquantitative and qualitative assays. The number of curves, theconfiguration and/or the length of the serpentine channel 96 may bedetermined based on the assay of interest.

FIGS. 6 and 7A-7C illustrate an exemplary method for operating theplasma separator system 10 of FIG. 1. In particular, FIG. 6 illustratesa flow chart 110 for operating the plasma separation system 10.

To separate plasma from red and white blood cells in a blood sample, ina step 112, the blood sample 130 (e.g., 100 μl) may be added to theblood separation well 14 as illustrated in FIG. 7A. In some embodiments,a pipet, or other similar device, may be used to add the blood sample tothe blood separation well 14.

In a step 114, the blood sample may wick into the filter 40 and theseparation membrane 42. In some embodiments, the blood sample may beallowed to wick into the filter 40 and/or the separation membrane 42 fora pre-determined time period. For example, in one non-limiting example,the blood sample may be allowed to wick into the filter 40 and/or theseparation membrane 42 for approximately 5-45 seconds.

In some embodiments, the blood sample may wick into the filter 40 andthe separation membrane 42 by capillary action. In one non-limitingexample, the presence of the one or more venting channels 68 (shown inFIG. 3) may promote wicking of the blood sample by providing escape ofgas from edges of the filter 40 and/or the separation membrane 42.Further, the presence of microchannels 64 on the capillary surface 38 ofthe recess 30 may also promote capillary flow from the second side 56 ofthe separation membrane 42 into the channel 16 (shown in detail in FIG.3).

In a step 116, the negative pressure source 26 (e.g., vacuum source) maybe actuated to apply the vacuum force to the blood separation well 14via the outlet port 24. In a step 118, the vacuum force assists theblood sample to proceed through the filter 40 providing filtered blood.In a step 120, the blood sample may proceed through the separationmembrane 42 providing filtered plasma 132 as illustrated in FIG. 7B. Itshould be noted that prior to application of vacuum source to the bloodseparation well 14, a portion of the blood sample may proceed throughthe filter 40 and/or the separation membrane 42.

In a step 122, the filtered plasma 132 may flow through the channel 16and collect in the plasma collection vessel 18 as illustrated in FIG.7C. The vacuum force may be maintained at a substantially constant leveluntil all needed filtered plasma is collected in the plasma collectionvessel 18 or may increase over time to a set-point.

In a step 124, the vacuum force is caused to cease, such as bydeactuating the negative pressure source 26 (e.g., vacuum source). In astep 126, the filtered plasma can be removed from the plasma collectionvessel 18 such as by piercing the seal 80 of the plasma collectionvessel 18. For example, a pipet tip may pierce the seal 80 and filteredplasma may be removed from the plasma collection vessel 18. One or moreassay may then be performed using the filtered plasma. Quantitativeand/or qualitative assays may be performed using the filtered plasma.For example, as the pipet may be capable of collecting a determinateamount of filtered plasma, quantitative assays may be performed.

In one example, a blood sample of 100 μL and 35% hematocrit (HCT)containing D-dimer at a concentration of 450 ng/ml may be added to theblood separation well 14 as illustrated in FIG. 7A. The filter 40 of theblood separation well 14 may be formed of VF2, and the separationmembrane 42 may be formed of a polysulfone asymmetric membrane, forexample. Using the process detailed in FIGS. 6 and 7, the blood samplemay be allowed to wick into the filter 40 and/or separation membrane 42for approximately 5-45 seconds. The negative pressure source 26 may beapplied (e.g., between 0.05-2 psi) such that filtered plasma (e.g.,approximately 20-25 μL) may be collected. The D-dimer concentration inthe filtered plasma may then be measured (e.g., using a Siemens StratusCS D-dimer immunoassay). In one example, the D-dimer concentrationrecovery for the filtered plasma, as compared to centrifugation was100.8%.

In another example, a blood sample of 100 μL and 42% HCT containing Tnlat a concentration of 100 pg/ml may be added to the blood separationwell 14. Using the process detailed in FIGS. 6 and 7, the blood samplemay be allowed to wick into the filter 40 and/or separation membrane 42for approximately 5-45 seconds. The negative pressure source 26 may beapplied (e.g., between 0.05-2 psi) such that filter plasma (e.g.,approximately 20 μL) may be collected. The Tnl concentration in thefiltered plasma may then be measured (e.g., using Siemens Dimension EXLTnl immunoassay). In one example, the Tnl concentration recovery for thefiltered plasma, as compared to centrifugation was 86%.

FIG. 8 illustrates another exemplary embodiment of a plasma separationsystem 10 c. The plasma separation system 10 c is similar to the plasmaseparation systems 10-10 b illustrated in FIGS. 1, 4 and 5 respectively;however, the plasma separation system 10 c applies positive force via apositive pressure source 140 upstream of the plasma collection vessel18. In particular, the positive pressure source 140 may be connected toa cap 142 positioned over the filter 40 within the blood separation well14.

In some embodiments, the cap 142 may be form fit over the filter 40within the blood separation well 14. The cap 142 may include an outlet144. The outlet 144 may connect to the positive pressure source 140. Thepositive pressure source 140 may provide between 0.05-2 psi of positivepressure to the blood separation well 14 during use. The positivepressure may force the blood sample through the filter 40, separationmembrane 42, and/or channel 16 into the plasma collection vessel 18.

In some embodiments, the positive pressure source 140 may be a syringeloaded with air. By forcing air through the outlet 144, positivepressure may be applied to the blood sample forcing the blood samplethrough the filter 40, separation membrane 42, and/or channel 16 to theplasma collection vessel 18. In this embodiment, the outlet 24downstream of the plasma collection vessel 18 may be used as a vent andopened as needed.

From the above description, it is clear that the inventive concept(s)disclosed herein are well adapted to carry out the objects and to attainthe advantages mentioned herein, as well as those inherent in theinventive concept(s) disclosed herein. While the embodiments of theinventive concept(s) disclosed herein have been described for purposesof this disclosure, it will be understood that numerous changes may bemade and readily suggested to those skilled in the art which areaccomplished within the scope and spirit of the inventive concept(s)disclosed herein.

The following is a list of non-limiting illustrative embodiments of theinvention:

1. An apparatus, comprising:

a blood separation well for collection of a blood sample, the bloodseparation well having a recess intersecting a surface of the bloodseparation well; a separation membrane positioned on the surface of therecess for filtration of the blood sample to provide filtered plasma; aplasma collection vessel; a first channel connecting the bloodseparation well to the plasma collection vessel proximate to the surfaceof the recess; and, a second channel connecting the plasma collectionvessel to a first outlet port, wherein the plasma collection vessel andthe first channel are configured to convey a pressure force to providefiltered plasma to the plasma collection vessel.

2. The apparatus of illustrative embodiment 1, wherein the pressureforce is a negative pressure force and the first channel, the plasmacollection vessel, and the second channel are configured to convey thenegative pressure force applied to the first outlet port to the surfaceof the recess.

3. The apparatus of illustrative embodiments 1 or 2, further comprisinga cap positioned on the separation membrane, the cap having a secondoutlet port formed therein, wherein the pressure is a positive pressureforce and the first channel is configured to convey the positivepressure force applied through the second outlet port to the plasmacollection vessel.

4. The apparatus of any one of illustrative embodiments 1-3, furthercomprising a filter positioned within the recess and on the separationmembrane, the filter having a plurality of structures surrounding poresconfigured to separate red blood cells from the blood sample to providefiltered blood, the filtered blood having a reduction in red blood cellsas compared to an amount of red blood cells in the blood sample.

5. The apparatus of illustrative embodiment 4, wherein at least one ofthe separation membrane or filter is coated with a blocking agent.

6. The apparatus of illustrative embodiment 4, wherein at least one ofthe separation membrane or filter is treated with a surfactant.

7. The apparatus of illustrative embodiment 4, further comprising a cappositioned at a surface of the housing for containing the separationmembrane and filter within the recess.

8. The apparatus of illustrative embodiment 7, wherein the cap includesat least one venting hole.

9. The apparatus of any one of illustrative embodiments 1-8, wherein thesurface is defined further as a capillary surface having at least onemicrochannel.

10. The apparatus of illustrative embodiment 9, wherein capillarysurface has a plurality of microchannels forming a concentric pattern inthe capillary surface, the concentric pattern having a set of radialmicrochannels projecting from a location.

11. The apparatus of any one of illustrative embodiments 1-10, furthercomprising a vacuum source connected to the second channel at the outletport.

12. The apparatus of illustrative embodiment 11, wherein the vacuumsource is a syringe pump, the syringe pump providing a vacuum forcebetween 0.05 psi and 2 psi.

13. The apparatus of any one of illustrative embodiments 1-13, whereinthe plasma collection vessel is a serpentine channel.

14. The apparatus of any one of illustrative embodiments 1-13, whereinthe plasma collection vessel has a proximal end and a distal end, thedistal end positioned at a surface of the housing and wherein the plasmacollection vessel includes a seal configured to be pierced by a pipette.

15. The apparatus of illustrative embodiment 14, wherein the firstchannel is connected to the plasma collection vessel at the proximal endof the plasma collection vessel.

16. The apparatus of any one of illustrative embodiments 1-15, whereinthe blood separation well further comprises at least one vent positionedin the recess of the blood separation well, the vent positioned beyondan outer edge of the separation membrane.

17. The apparatus of any one of illustrative embodiments 1-16, furthercomprising an adhesive member connecting the surface and the separationmembrane within the recess.

18. The apparatus of any one of illustrative embodiments 1-17, whereinthe separation membrane is an asymmetric membrane having a first set ofpores larger than a second set of pores, the first set of pores locatedon a first side of the separation membrane and the second set of poreslocated on a second side of the separation membrane.

19. The apparatus of any one of illustrative embodiments 1-18, whereinthe separation membrane is pressure fit within the recess of the bloodseparation well.

20. The apparatus of any one of illustrative embodiments 1-19, whereinthe plasma collection vessel is conically shaped and has capacity tocollect at least 20 μL of filtered plasma.

21. The apparatus of any one of illustrative embodiments 1-20, furthercomprising a housing having a surface, wherein the recess of the bloodseparation well intersects the surface of the housing and the surface ofthe blood separation well.

22. A kit comprising: a plasma separation system comprising: a housinghaving a surface; a blood separation well for collection and filtrationof a blood sample to provide filtered plasma, the blood separation wellhaving a recess intersecting the surface of the housing and a surface ofthe blood separation well;

an adhesive member positionable on the surface of the recess; aseparation membrane configured to adhere to the adhesive member; and; afilter positionable on the separation membrane and pressure fit withinthe recess; a plasma collection vessel; a first channel connecting thesurface of the blood separation well to the plasma collection vessel; asecond channel connecting the plasma collection vessel to an outletport; and, a vacuum source configured to connect to the outlet port, thevacuum source configured to provide between 0.05 psi and 2 psi vacuumforce to the outlet port.

23. A method comprising: providing a blood sample to a plasma separationsystem, the plasma separation system comprising: a blood separation wellcontaining a filter and a separation membrane, the blood separation wellconnected to a plasma collection vessel by a first channel, and theplasma collection vessel connected to a second channel with an outletport, the outlet port connected to a vacuum source; wherein the bloodsample wicks into the filter for a first pre-determined time interval;and, actuating the vacuum source to apply a vacuum source to the bloodseparation well via the outlet port to enhance flow of plasma from theblood sample through the filter and separation membrane providingfiltered plasma, and to draw the filtered plasma through the firstchannel into the plasma collection vessel.

24. The method of illustrative embodiment 23, wherein the plasmacollection vessel includes a seal; the method further comprising:piercing, with a pipette, the seal of the plasma collection vessel; and,collecting filtered plasma with the pipette.

What is claimed is:
 1. A kit comprising: a plasma separation systemcomprising: a housing having a first surface; a blood separation wellformed at the first surface of the housing for collection and filtrationof a blood sample to provide filtered plasma, the blood separation wellhaving a recess intersecting a second surface of the housing within theblood separation well; an adhesive member positionable on the secondsurface of the housing within the blood separation well; a separationmembrane configured to adhere to the adhesive member; a filterpositionable on the separation membrane; a plasma collection vesselwithin the housing, the plasma collection vessel having a proximal endand a distal end; a first channel extending within the housing andconnecting the blood separation well to the plasma collection vessel; asecond channel connecting the plasma collection vessel to an outletport; and, a vacuum source configured to connect to the outlet port, thevacuum source configured to provide between 0.05 psi and 2 psi vacuumforce to the outlet port.
 2. The kit of claim 1, wherein the firstchannel, the plasma collection vessel, and the second channel areconfigured to convey the vacuum force applied to the outlet port to therecess.
 3. The kit of claim 1, further comprising a cap positioned onthe separation membrane, the cap having a second outlet port formedtherein, wherein the first channel is configured to convey a positivepressure force applied through the second outlet port to the plasmacollection vessel.
 4. The kit of claim 1, wherein the filter has aplurality of structures surrounding pores configured to separate redblood cells from the blood sample to provide filtered blood, thefiltered blood having a reduction in red blood cells as compared to anamount of red blood cells in the blood sample.
 5. The kit of claim 4,wherein at least one of the separation membrane or filter is coated witha blocking agent.
 6. The kit of claim 4, wherein at least one of theseparation membrane or filter is treated with a surfactant.
 7. The kitof claim 1, wherein the second surface is defined further as a capillarysurface having at least one microchannel.
 8. The kit of claim 7, whereinthe capillary surface has a plurality of microchannels forming aconcentric pattern in the capillary surface, the concentric patternhaving a set of radial microchannels projecting from a location.
 9. Thekit of claim 1, wherein the vacuum source is a syringe pump.
 10. The kitof claim 1, wherein the first channel is connected to the plasmacollection vessel at the proximal end of the plasma collection vessel.11. The kit of claim 1, wherein the blood separation well furthercomprises at least one vent positioned in the recess of the bloodseparation well, the vent positioned beyond an outer edge of theseparation membrane.
 12. The kit of claim 1, wherein the separationmembrane is an asymmetric membrane having a first set of pores largerthan a second set of pores, the first set of pores located on a firstside of the separation membrane and the second set of pores located on asecond side of the separation membrane.
 13. The kit of claim 1, whereinthe housing is a unitary integral device that is shaped to form theblood separation well, the plasma collection vessel, the first channel,and the second channel.
 14. The kit of claim 1, wherein the firstchannel intersects the plasma collection vessel a distance away from thedistal end of the plasma collection vessel.
 15. The kit of claim 1,wherein the first channel intersects the plasma collection vesselbetween the proximal end and the distal end.
 16. The kit of claim 1,wherein the first channel intersects the proximal end of the plasmacollection vessel.